This method is suitable for determining and confirming the presence of flunixin in bovine and porcine liver and muscle.

Method Summary

Homogenized tissue is hydrolized with HCl in tubes incubated for 2 hours on a heating block set between 95 -120 degrees Celsius before cooling the mixture to ambient temperature. The pH of the hydrolysate is adjusted to 9.5 (9.30 - 9.70) using NaOH. Ethyl
acetate is added to the tube prior to extraction by repeated centrifugation. The upper ethyl acetate extract is then loaded onto SCX cartridges for clean up and the analyte is eluted with methanolic ammonium hydroxide solution. This solution is evaporated
to dryness and the residue reconstituted in 50 percent methanol/water. The mixture is then pressed through 0.45 micometer PTFE filters ready for analysis by LC/ESI/MSMS. An Eclipse XDB-C18 analytical column is used

Applicable Concentration Range

Flunixin may be detected/quantified in bovine liver and muscle at ≥ 62.5 µg kg-1 and at ≥ 12.5 µg kg-1 concentration levels, respectively, and in porcine liver and muscle at ≥ 15.0 ppb and at ≥ 12.5 ppb, respectively